Polyketides, which are compounds synthesized from 2-carbon units by the action of polyketide synthases through a series of condensations and subsequent modifications, are produced in several bacteria, including molds or actinomycetes (Robinson, J. A., Philos. Trans. R. Soc. Lond. B. Biol. Sci., 332:107-14, 1991; Hopwood, D. A., Curr. Opin. Biotechnol., 4:531-537, 1993). The polyketides are physiologically active molecules having highly various structures and the species thereof includes many compounds having various activities.
Generally, a production method using such natural polyketides has encountered a difficulty in industrial application because of its low efficiency, and also faced many economic and technical difficulties in producing polyketide compounds by the prior chemical method.
Owing to such concerns, studies to efficiently biosynthesize the natural polyketides were conducted, and a recombinant production method using related genes started to be developed. Namely, studies, including cloning, assay and operation using a recombinant technology of genes coding for polyketide synthases, were performed, and owing to such technologies, the production of polyketides at a higher level than those occurring in an environment or host, which does not produce polyketides, became possible (WO 93/13663; 95/08548; 96/40968; 97/02358; 98/27203; 98/49315; U.S. Pat. Nos. 4,874,748, 5,063,155; 5,098,837; 5,149,639; 5,672,491; 5,712,146; 5,830,750; 5,843,718; Fu, H. et al., Biochem., 33:9321-6, 1994; McDaniel, R. et al., Science, 262:1546-50, 1993; Kuhstoss, S. et al., Gene, 183:231-6, 1996).
The natural polyketides is synthesized by the continuous activity of about 50 species of enzymes, so-called polyketide synthases (PKSs), and carrier proteins, in a similar manner as the synthesis of fatty acids (Robinson, J. A., Philos. Trans. R. Soc. Lond. B. Biol. Sci., 332:107-14, 1991; Hopwood, D. A., Curr. Opin. Biotechnol., 4:531-7, 1993). For example, the core structure of erythromycin is made by about 25 continuous enzymatic reactions. PKSs include two types, one of which is a modular type and the other one is an iteractive type (Khosla, C., Chem. Rev., 97:2577-90, 1997).
In the modular PKSs (type I), all steps for the assembly and modification of carbon chains occur by different catalytic sites, but in the iteractive PKSs (type II), one catalytic site is utilized one or more times on biosynthetic pathway. A typical modular PKS consists of several large peptides, which are divided into a loading module, multiple extension modules and a releasing module in the direction from the N-terminal end to the C-terminal end (Hopwood and Sherman, Annu. Rev. Genet., 24:37-66, 1990).
The loading module consists of acyl-transferase (AT) and acyl carrier protein (ACP). Furthermore, the multiple extension modules basically comprise ketosynthase (KS), AP and ACP, and often comprise an enzyme for modifying the β-carbon of the extended polyketide chain in addition to such basic modules. Finally, the releasing module comprises thioesterase (TE) and in some cases, cyclase activity.
Generally, the loading module binds to a first building block used in synthesizing polyketides, and acts to transfer the building block to the first extension module. AT on the multiple extension modules recognize a certain acyl-CoA (acetyl or propionyl) and transfer it to ACP in the form of thiol ester. At the same time, AT on each module transfers the malonyl group of a certain malonyl-CoA (malonyl or α-substituted malonyl) to ACP on the module in a thiol ester form. Then, the acyl group on the loading module is transferred to KS on the first module by transesterification. The transferred acyl group covalently binds to the α-carbon of the malonyl group, and dicarboxylation occurs to produce a new acyl group with a backbone two carbons longer than the loaded unit (extension or elongation).
The polyketide chain grown by two carbon atoms of each extension module is transferred from the extension module to the next extension module in the form of covalently bonded thiol ester, and subjected to the above-mentioned procedure. In addition to KS, AT and ACP involved in the formation of the C—C bonds, each module often comprises enzymes for modifying the β-keto group of 2-carbon units just added to the extended polyketide chain before this chain is transferred to the next module. These modifier enzymes include ketoreductase (KR) reducing a keto group into alcohol, dehydroreductase (DH) forming a double bond by dehydration of alcohol, and enoyl reductase (ER) converting a double bond into a single bond.
There are modules having no modifier enzymes, and modules comprising KR(1), KR+DH(2) or KR+DH+ER(3). According to such modifier enzymes, the oxidation state of the β-carbon of each 2-carbon unit is determined (0=ketone; 1=alcohol; 2=double bond; and 3=single bond). Polyketide products will vary depending on the specificity of AT of each module and the kind of the modifier enzymes. If the extended polyketide chain is transferred to the last module of PKS, it will meet the releasing module or thioesterase active site in which polyketide is truncated into a ring form. Furthermore, polyketide can be further modified by adding a carbohydrate or methyl group to the core molecule thereof or by a tailoring enzyme (TE) inducing other modifications.
As described above, polyketides are formed by the condensation of carboxylic acid units using the continuous action of PKS. Most of PKS genes in microorganisms are known as being present in a cluster form in the microbial chromosome (Hopwood, Chem. Rev., 97:2465-97, 1997). In 1960, DEBS genes involved in the formation of 6-deoxyerythronolide B (6 dEB) were first reported (Cortes et al., Nature, 348:176-8, 1990; Donadio et al., Science, 252:675-9, 1991), and then, the cloning and base sequence of whole or partial genes for the synthesis of eleven polyketides were reported up to now. The genetic information of the polyketides reported up to now is summarized in Table 1 below.
TABLE 1Genetic information of polyketides reported up to now.Open Reading FramesPolyketideSize(ORFs)ReferencesAvermectin80 kb18 ORFs (containing 4 PKSes)U.S. Pat. No. 5,252,474; Ikeda, et al., PNAS USA, 96: 9509-14, 1999FK50660 kb 6 ORFs (containing 3 PKSes)Motamedi & Shafiee, Eur. J. Biochem., 256: 528-34, 1998Rapamycin110 kb 26 ORFs (containing 3 PKSes)Schwecke, et al., PNAS USA., 92: 7839-43, 1995Rifampicin90 kb34 ORFs (containing 5 PKSes)August, et al., Chem. Biol., 5: 69-79, 1998Tylosin85 kb41 ORFs (containing 5 PKSes)Cundliffe, et al., Ant. van Leeuwen., 79: 229-34, 2001Nystatin124 kb 22 ORFs (containing 6 PKSes)Brautaset, et al., Chem. Biol., 7: 395-403, 2000Pimaricin85 kb17 ORFs (containing 5 PKSes)Aparicio, et al., Chem. Biol., 7: 895-905, 2000Amphotericin113 kb 17 ORFs (containing 6 PKSes)Caffrey, et al., Chem. Biol., 8: 713-23, 2000Candicidin D70 kb16 ORFs (containing 4 PKSes)Campelo & Gil, Microbiol., 148: 51-9, 2002(partial)
FR-008 polyketide produced in Streptomyces sp. FR-008 is a heptaene macrolide having aglycone containing 4-aminoacetophenone, as in candicidin D (Yuan and Zhou, J. Huazhong Agricult. Univ., 9:209, 1990). Since the FR-008 polyketide has antifungal activity and also high toxicity against mosquito larva, they are highly useful in agricultural and medical fields (Liang and Zhou, Chinese J. Biotech., 3:130-6, 1987). The FR-008 polyketide has the following formula:

Genes for the synthesis of the FR-008 polyketide was first cloned in 1994 (Hu et al., Mol. Microbiol., 14:163-72, 1994). Hu et al. constructed a chromosomal library of Streptomyces sp. FR-008 using a cosmid vector and screened 16 cosmid clones containing PKS genes from the same, but the base sequence of the genes was not reported.